To identify this molecule, tryptic peptide digests were analyzed by Nano liquid chromatography-mass spectrometry (MS)
To identify this molecule, tryptic peptide digests were analyzed by Nano liquid chromatography-mass spectrometry (MS). an antibacterial activity (Figure 1b). Open in a separate window Figure 1 Purification of the antimicrobial activity from extracts of cultured sebocytes(a) RP-HPLC was carried out to separate antimicrobial peptides from acid-soluble extracts of SEB-1 sebocytes. A plot of absorbance at 214nm is indicated on the left axis and that of the concentration of acetonitrile used for elution on the right axis. (b) The antimicrobial activity of the HPLC fractions was evaluated against by solution-killing assay. Growth of bacterial colonies on agar after incubation with fractions obtained by RP-HPLC separation is shown with limiting dilutions from top to bottom. A single fraction (fraction 13) showed maximal antimicrobial activity. (c) HPLC fractions were subjected to SDS-PAGE and stained with Coomassies blue. Arrow in the active fraction indicates a unique protein band that was further analyzed by mass spectrometry. Identification of Histone H4 as an antimicrobial peptide As predicted by the initial Alfuzosin HCl HPLC tracing, each of the fractions separated from sebocyte extracts contained multiple protein bands (Figure 1c). On the basis of the findings of the antimicrobial activity and the SDS-PAGE, one band migrating at approximately 11 kDa in the active fraction was found to be interesting. To identify this molecule, tryptic peptide digests were analyzed by Nano liquid chromatography-mass spectrometry (MS). Histones, H2A, H2B, and Alfuzosin HCl H4, were identified with H4 being most abundant (Table 1). Table 1 Proteins identified by mass spectrometry from sebocyte antimicrobial fraction and or (25 g ml?1) and (12.5 g ml?1) in a dose-dependent manner compared with the control (Figure 3). Furthermore, as histone H4 is released from sebocytes along with lipids, which are metabolized into free fatty acids with antimicrobial activity, the combined antimicrobial activity of histone H4 in the presence of free fatty acids was examined. Lauric acid and oleic acid at low concentrations (1.5 g ml?1) enhanced the antimicrobial action of histone H4 (Figure 4). At higher concentrations of fatty acids (60 g ml?1), lauric acid completely inhibited survival at 12.5 g ml?1 histone H4, but oleic acid at this concentration killed all of the bacteria. Thus, as expected, free fatty acids present on the skin are inherently antimicrobial, but will also enhance the antimicrobial potency of peptides such as histone H4 released by the sebocyte. Open Rabbit Polyclonal to MAP3KL4 in a separate window Figure 3 Recombinant histone H4 kills and and by the solution-killing assay. Bacteria were incubated for 3 hours at 37C in a 10mm sodium phosphate buffer (pH 6.5) containing 100mM NaCl. Recombinant histone H4 showed significant bactericidal activity against at 25 g ml?1 and above and against at 12.5 g ml?1 and above. Data shown are from triplicate Alfuzosin HCl determinations (**as seen by an approximately two logs better growth of bacteria in the presence of a sebocyte extract plus BWA-3 compared with that with a sebocyte extract plus IgG at a similar concentration, or with PBS as controls (Figure 5). Thus, the ability to neutralize the majority of the antimicrobial action of sebocyte extracts with a specific antibody to histone H4 suggests that histone H4 is the major acid-soluble antimicrobial protein released by the sebocyte. Open in a separate window Figure 5 Neutralization of histone H4 in sebocyte extracts removes antimicrobial activityBWA-3 neutralizing antibody was used to treat extracts of SEB-1 sebocytes. Addition of BWA-3 (400 g ml?1) to the extracts of SEB-1 sebocytes significantly decreased antimicrobial activity against compared with an equal concentration of IgG added to separate aliquots, or aliquots Alfuzosin HCl to which an equal volume of PBS was added as controls. Data shown are from triplicate determinations (*and (Rose is released with sebum onto the surface of the skin, and once released can act as an antimicrobial. As major components of the nucleosome structure in eukaryotic cells, the histones are functionally classified into two groups: linker histones (H1), which seal loops of DNA and keep the nucleosome structures condensed in compact conformations, and core histones (H2A, H2B, H3, and H4), which form an octameric complex to produce the nucleosome. Most earlier studies have shown that the lysine-rich histones Alfuzosin HCl (H1, H2A, and H2B) and their fragments have antimicrobial activity (Hiemstra and studies of the surface expression of histones on human skin, and mechanistic studies to determine how histone H4 acts as an antimicrobial peptide, are.